The p53 gene is the most commonly mutated tumor suppressor with inactivating mutations in over 50% of all human cancers. The remaining half of cancers contain wild-type p53, but harbor additional means of p53 inactivation. Recent cancer therapies focused on restoring p53's tumor suppressive program include the small molecule inhibitor Nutlin-3, which activates dormant p53 through inhibiting the interaction between p53 and its endogenous repressor MDM2. Importantly, this non-genotoxic approach would replace toxic chemotherapy treatments, which carry harmful side-effects by activating p53 through means of DNA damage. Despite the considerable momentum with Nutlin-3, its efficacy is currently limited by the heterogeneity of the p53 response, where most cancer cells undergo reversible cell cycle arrest, as opposed to a therapeutically favorable apoptotic response. In order to improve p53-based therapies, a better understanding of the mechanisms governing p53-induced apoptosis is needed. Additionally, previous studies demonstrate that p53s tumor suppressive function is dependent on its role as a transcription factor, yet no direct p53 target t date has been identified as crucial to p53-mediated tumor suppression. The objectives herein are to identify the cellular factors necessary to tip the balance of the p53 response toward apoptosis, and to determine what direct p53 transcriptional targets are necessary for p53 mediated cell death. To answer these questions the following specific aims will be developed: 1) Identify novel mediators of p53-dependent apoptosis in response to non- genotoxic activation of p53 by Nutlin-3, 2) Identify direct transcriptional targets necessary for p53-mediated cell death, and 3) Determine the impact of novel mediators of p53-dependent apoptosis on tumor suppression in vivo. In answering these questions, a powerful functional genomics approach will be applied in two pediatric cancer cell lines, which have been identified as having strong apoptotic responses to Nutlin-3. For an unbiased approach to identify novel mediators of p53-dependent apoptosis, a positive selection screen with a genome wide shRNA library will be utilized, and validation of top candidates will clearly identify genes that, when knocked down, confer resistance to Nutlin treatment. For direct p53 transcriptional targets essential for cell death, a similar positive selection screen will be conducted, only now with a uniquely comprised focused P53TARGET shRNA library, targeting ~300 direct p53 targets. After validation of top candidates, investigations into the mechanism by which these genes mediate p53-dependent apoptosis will be conducted. To translate these findings into clinical relevance, the identified apoptotic regulators will also be tested for their ability to mediate p53-dependent cell death in a tumor xenograft (in vivo) model. Results herein will illuminate the cellular factors essential for p53-mediated cell death, which is vital for the improvement of p53-reactivation strategies. Once effective, this treatment option will be available to all cancers that retain wild-type p53, nearly11 million people worldwide.